1. Field of the Invention
The present invention relates to a sensor that is used in sensing, in which a substance to be detected is marked with a fluorescent marker that selectively binds to the substance to be detected and one of two-photon excitation fluorescence and multi-photon excitation fluorescence of the fluorescent marker is detected. The present invention also relates to a sensing apparatus and a sensing method using the sensor.
2. Description of the Related Art
In recent years, fluorescence measurement methods have been proposed in fluorescent analysis that is performed in immunity measurement and the like in the field of biochemistry. In the fluorescence measurement methods, a sensor having a sensing surface, to which a specific substance to be detected can bind, is used. In the methods, a fluorescent marker that has bound to the substance to be detected is excited by evanescent waves or surface plasmons and the substance to be detected is sensed.
In such methods, fluorescence is emitted only when the substance to be detected has bound to the sensing surface. Therefore, real-time measurement is possible. Further, since excitation light does not reach a detector, background light of fluorescence, the fluorescence that should be detected, is small. Therefore, it is possible to perform measurement at a relatively high S/N ratio.
However, in the fluorescence measurement method, substances, such as water, blood serum protein and an enzyme, which coexist with the substance to be detected in a sample, may absorb excitation light. Further, some substance may emit unwanted fluorescence. Further, when surface plasmons are used to excite the fluorescent marker, the substance to be detected needs to be bound to a sensor that is formed by a prism, the surface of which is coated with a thin metal film that generates surface plasmons. If such a sensor is used, the prism absorbs excitation light and emits unwanted fluorescence in some cases. Such absorption of excitation light and emission of fluorescence by coexisting substances or the like, which are not an object of detection, lower the accuracy of detection of fluorescence that should be originally detected.
As a method for performing fluorescence measurement at a higher S/N ratio by suppressing absorption of excitation light and emission of fluorescence by coexisting substances or the like, a two-photon excitation fluorescence measurement method has been proposed. In the two-photon excitation fluorescence measurement method, a fluorescent marker that can be excited by two-photon excitation is used. Two-photon excitation fluorescence is generated by using excitation light that has twice the wavelength of excitation light that is used to generate ordinary fluorescence. Therefore, in the two-photon excitation fluorescence measurement method, it is possible to use excitation light that has a wavelength different from the absorption wavelength of a coexisting substance or the like. Hence, it is possible to perform low-noise fluorescent measurement. Further, since excitation light that has a wavelength in a near-infrared band is used, even if the substance to be detected is living-body tissue, there is no risk that the substance to be detected is damaged by measurement.
However, the cross-sectional area of absorption by two-photon excitation is smaller than that of absorption by single-photon excitation by several tens of digits. Therefore, ordinarily, a very expensive pulse laser that has a high peak value is used to obtain sufficient fluorescence. As a method for obtaining sufficient fluorescence without using such a pulse laser, a method for exciting two-photon excitation fluorescence by surface plasmons has been disclosed (Japanese Unexamined Patent Publication No. 2001-021565).
In Japanese Unexamined Patent Publication No. 2001-021565, features that the probability of transition is increased by at least two digits by exciting two-photon excitation fluorescence by an electric field generated by surface plasmons and the fluorescence efficiency is remarkably improved are described. However, when the surface plasmons are utilized, a sensor formed by a prism that is coated with a thin metal film that generates surface plasmons is required. Further, a complicated optical system is required to emit measurement light and to detect detection light. Therefore, the sensing apparatus tends to be expensive and complicated. Further, even if the probability of transition is increased by approximately two digits, when the size of the cross-sectional area of absorption is taken into consideration, it is not recognized that sufficient fluorescence efficiency is obtained. Therefore, there is still a problem that the fluorescence efficiency has to be improved.